Human vascular endothelial growth factor 2

ABSTRACT

Disclosed is a human VEGF2 polypeptide and DNA (RNA) encoding such VEGF2 polypeptides. Also provided is a procedure for producing such polypeptide by recombinant techniques and antibodies and antagonist against such polypeptide. Also disclosed is a method of using such polypeptide for stimulating wound healing and for vascular tissue repair. Also provided are methods of using the antagonists to inhibit tumor growth, inflammation and to treat diabetic retinopathy, rheumatoid arthritis and psoriasis. Diagnostic methods for detecting mutations in the VEGF2 coding sequence and alterations in the concentration of VEGF2 protein in a sample derived from a host are also disclosed.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation, and claims benefit ofpriority under 35 U.S.C. §120, of U.S. application Ser. No. 08/465,968,filed Jun. 6, 1995, currently suspended, which is a contiuation-in-partof of U.S. application Ser. No. 08/207,550, file Mar. 8, 1994, nowabandoned, both of which are herein incorporated by reference in theirentirety.

SUMMARY OF THE INVENTION

[0002] This invention relates to newly identified polynucleotides,polypeptides encoded by such polynucleotides, the use of suchpolynucleotides and polypeptides, as a well as the production of suchpolynucleotides and polypeptides. The polypeptide of the presentinvention has been identified as a member of the vascular endothelialgrowth factor family. More particularly, the polypeptide of the presentinvention is vascular endothelial growth factor 2, sometimes hereinafterreferred to as “VEGF-2.” The invention also relates to inhibiting theaction of such polypeptide.

BACKGROUND OF THE INVENTION

[0003] The formation of new blood vessels, or angiogenesis, is essentialfor embryonic development, subsequent growth, and tissue repair.Angiogenesis, however, is an essential part of certain pathologicalconditions such as neoplasia, for example, tumors and gliomas, andabnormal angiogenesis is associated with other diseases such asinflammation, rheumatoid arthritis, psoriasis, and diabetic retinopathy(Folkman, J. and Klagsbrun, M., Science 235:442-447,(1987)).

[0004] Both acidic and basic fibroblast growth factor molecules aremitogens for endothelial cells and other cell types. Angiotropin andangiogenin can induce angiogenesis, although their functions are unclear(Folkman, J., 1993, Cancer Medicine pp. 153-170, Lea and Febiger Press).A highly selective mitogen for vascular endothelial cells is vascularendothelial growth factor or VEGF (Ferrara, N., et al., Endocr. Rev.13:19-32, (1992)), also known as vascular permeability factor (VPF).Vascular endothelial growth factor is a secreted angiogenic mitogenwhose target cell specificity appears to be restricted to vascularendothelial cells.

[0005] The murine VEGF gene has been characterized and its expressionpattern in embryogenesis has been analyzed. A persistent expression ofVEGF was observed in epithelial cells adjacent to fenestratedendothelium, e.g., in choroid plexus and kidney glomeruli. The data wasconsistent with a role of VEGF as a multifunctional regulator ofendothelial cell growth and differentiation (Breier, G. et al.Development, 114:521-532 (1992)).

[0006] VEGF is structurally related to the and chains ofplatelet-derived growth factor (PDGF), a mitogen for mesenchymal cellsand placenta growth factor (PLGF), an endothelial cell mitogen. Thesethree proteins belong to the same family and share a conserved motif.Eight cysteine residues contributing to disulfide-bond formation arestrictly conserved in these proteins. Alternatively spliced mRNAs havebeen identified for VEGF, PLGF and PDGF and these different splicingproducts differ in biological activity and in receptor-bindingspecificity. VEGF and PDGF function as homo-dimers or hetero-dimers andbind to receptors which elicit intrinsic tyrosine kinase activityfollowing receptor dimerization.

[0007] VEGF has four different forms of 121, 165, 189 and 206 aminoacids due to alternative splicing. VEGF121 and VEGF165 are soluble andare capable of promoting angiogenesis, whereas VEGF189 and VEGF-206 arebound to heparin containing proteoglycans in the cell surface. Thetemporal and spatial expression of VEGF has been correlated withphysiological proliferation of the blood vessels (Gajdusek, C. M., andCarbon, S. J., Cell Physiol., 139:570-579, (1989)); McNeil, P. L.,Muthukrishnan, L., Warder, E., D' Amore, P. A., J. Cell. Biol.,109:811-822, (1989)). Its high affinity binding sites are localized onlyon endothelial cells in tissue sections (Jakeman, L. B., et al., Clin.Invest. 89:244-253, (1989)). The factor can be isolated from pituitarycells and several tumor cell lines, and has been implicated in somehuman gliomas (Plate, K. H. Nature 359:845-848, (1992)). Interestingly,expression of VEGF121 or VEGF165 confers on Chinese hamster ovary cellsthe ability to form tumors in nude mice (Ferrara, N., et al., J. Clin.Invest. 91:160-170, (1993)). The inhibition of VEGF function byanti-VEGF monoclonal antibodies was shown to inhibit tumor growth inimmune-deficient mice (Kim, K. J., Nature 362:841-844, (1993)). Further,a dominant-negative mutant of the VEGF receptor has been shown toinhibit growth of glioblastomas in mice.

[0008] Vascular permeability factor, has also been found to beresponsible for persistent microvascular hyperpermeability to plasmaproteins even after the cessation of injury, which is a characteristicfeature of normal wound healing. This suggests that VPF is an importantfactor in wound healing. Brown, L. F. et al., J. Exp. Med., 176:1375-9(1992).

[0009] The expression of VEGF is high in vascularized tissues, (e.g.,lung, heart, placenta and solid tumors) and correlates with angiogenesisboth temporally and spatially. VEGF has also been shown to induceangiogenesis in vivo. Since angiogenesis is essential for the repair ofnormal tissues, especially vascular tissues, VEGF has been proposed foruse in promoting vascular tissue repair (e.g., in atherosclerosis).

[0010] U.S. Pat. No. 5,073,492, issued Dec. 17, 1991 to Chen et al.,discloses a method for synergistically enhancing endothelial cell growthin an appropriate environment which comprises adding to the environment,VEGF, effectors and serum-derived factor. Also, vascular endothelialcell growth factor. C sub-unit DNA has been prepared by polymerase chainreaction techniques. The DNA encodes a protein that may exist as eithera hetero-dimer or homo-dimer. The protein is a mammalian vascularendothelial cell mitogen and, as such, is useful for the promotion ofvascular development and repair, as disclosed in European PatentApplication No. 92302750.2, published Sep. 30, 1992.

[0011] The polypeptides of the present invention have been putativelyidentified as a novel vascular endothelial growth factor based on aminoacid sequence homology to human VEGF.

DETAILED DESCRIPTION OF THE INVENTION

[0012] In accordance with one aspect of the present invention, there areprovided novel mature polypeptides, as well as biologically active anddiagnostically or therapeutically useful fragments, analogs andderivatives thereof. The polypeptides of the present invention are ofhuman origin.

[0013] In accordance with another aspect of the present invention, thereare provided isolated nucleic acid molecules encoding the polypeptidesof the present invention, including mRNAs, DNAs, cDNAs, genomic DNA aswell as biologically active and diagnostically or therapeutically usefulfragments, analogs and derivatives thereof.

[0014] In accordance with still another aspect of the present invention,there are provided processes for producing such polypeptides byrecombinant techniques comprising culturing recombinant prokaryoticand/or eukaryotic host cells, containing a nucleic acid sequenceencoding a polypeptide of the present invention, under conditionspromoting expression of said proteins and subsequent recovery of saidproteins.

[0015] In accordance with yet a further aspect of the present invention,there is provided a process for utilizing such polypeptide, orpolynucleotide encoding such polypeptide for therapeutic purposes, forexample, to stimulate angiogenesis, wound-healing, and to promotevascular tissue repair.

[0016] In accordance with yet another aspect of the present invention,there are provided antibodies against such polypeptides.

[0017] In accordance with yet another aspect of the present invention,there are provided antagonists to such polypeptides, which may be usedto inhibit the action of such polypeptides, for example, to inhibit thegrowth of tumors, to treat diabetic retinopathy, inflammation,rheumatoid arthritis and psoriasis.

[0018] In accordance with another aspect of the present invention, thereare provided nucleic acid probes comprising nucleic acid molecules ofsufficient length to specifically hybridize to nucleic acid sequences ofthe present invention.

[0019] In accordance with another aspect of the present invention, thereare provided methods of diagnosing diseases or a susceptibility todiseases related to mutations in nucleic acid sequences of the presentinvention and proteins encoded by such nucleic acid sequences.

[0020] In accordance with yet a further aspect of the present invention,there is provided a process for utilizing such polypeptides, orpolynucleotides encoding such polypeptides, for in vitro purposesrelated to scientific research, synthesis of DNA and manufacture of DNAvectors.

[0021] These and other aspects of the present invention should beapparent to those skilled in the art from the teachings herein.

[0022] The following drawings are illustrative of embodiments of theinvention and are not meant to limit the scope of the invention asencompassed by the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

[0023]FIG. 1A-1C shows the cDNA sequence and the corresponding deducedamino acid sequence of the polypeptide of the present invention. Thestandard one-letter abbreviations for amino acids are used. Sequencingwas performed using 373 Automated DNA Sequencer (Applied Biosystems,Inc.). Sequencing accuracy is predicted to be greater than 97%.

[0024]FIG. 2A-2B is an illustration of the amino acid sequence homologybetween the polypeptide of the present invention and other members ofthe human PDGF/VEGF family. The boxed areas indicate the conservedsequences and the location of the eight conserved cysteine residues. Theamino acid sequences shown in FIG. 2A-2B at comparative lines 1-3 (SEQID NOS: 3-5, respectively) and the fourth line amino acid sequence fromSEQ ID NO: 2 (corresponding to amino acids 24 to 373) are represented bythe one-letter amino acid codes.

[0025]FIG. 3 shows a photograph of a gel after in vitro transcription,translation and electrophoresis of the polypeptide of the presentinvention. Lane 1: ¹⁴C and rainbow M.W. marker; Lane 2: FGF control;Lane 3: VEGF-2 produced by M13-reverse and forward primers; Lane 4:VEGF-2 produced by M13 reverse and VEGF-F4 primers; Lane 5: VEGF-2produced by M13 reverse and VEGF-F5 primers.

[0026]FIG. 4A-4B. VEGF-2 polypeptide is expressed in a baculovirussystem consisting of Sf9 cells. Protein from the medium and cytoplasm ofcells were analyzed by SDS-PAGE under reducing and non-reducingconditions.

[0027]FIG. 5. The medium from Sf9 cells infected with a nucleic acidsequence of the present invention was precipitated and the resuspendedprecipitate was analyzed by SDS-PAGE and was stained with coomassiebrilliant blue.

[0028]FIG. 6. VEGF-2 was purified from the medium supernatant andanalyzed by SDS-PAGE in the presence or absence of the reducing agent-mercaptoethanol and stained by coomassie brilliant blue.

[0029]FIG. 7. Reverse phase HPLC analysis of purified VEGF-2 using aRP-300 column (0.21×3 cm, Applied Biosystems, Inc.). The column wasequilibrated with 0.1% trifluoroacetic acid (Solvent A) and the proteinseluted with a 7.5 min gradient from 0 to 60% Solvent B, composed ofacetonitrile containing 0.07% TFA. The protein elution was monitored byabsorbance at 215 nm (Red line) and 280 nm (Blue line). The percentageof Solvent B is shown by Green line.

[0030]FIG. 8 illustrates the effect of partially purified VEGF-2 proteinon the growth of vascular endothelial cells in comparison to basicfibroblast growth factor.

[0031]FIG. 9 illustrates the effect of purified VEGF-2 protein on thegrowth of vascular endothelial cells.

[0032] The term “gene” means the segment of DNA involved in producing apolypeptide chain; it includes regions preceding and following thecoding region (leader and trailer), as well as intervening sequences(introns) between individual coding segments (exons).

[0033] In accordance with one aspect of the present invention, there areprovided isolated nucleic acid molecules (polynucleotides) which encodefor the mature polypeptides having the deduced amino acid sequence ofFIG. 1A-1C or for the mature polypeptide encoded by the cDNA of theclone deposited as ATCC Deposit No. 97149 on May 12, 1995 or forpolypeptides which have fewer amino acid residues than those showing inFIG. 1A-1C. This deposit is a biological deposit made with the ATCC,10801 University Boulevard, Manassas, Va. 20110-2209 on the above date.

[0034] A polynucleotide encoding a polypeptide of the present inventionmay be obtained from early stage human embryo (week 8 to 9)osteoclastomas, adult heart or several breast cancer cell lines. Thepolynucleotide of this invention was discovered in a cDNA libraryderived from early stage human embryo week 9. It is structurally relatedto the VEGF/PDGF family. VEGF-2 contains an open reading frame encodinga protein of 419 amino acid residues of which approximately the first 23amino acid residues are the putative leader sequence such that themature protein comprises 396 amino acids, and which protein exhibits thehighest amino acid sequence homology to human vascular endothelialgrowth factor (30% identity), followed by PDGFα (23%) and PDGFβ (22%).

[0035] It is particularly important that all eight cysteines areconserved within all four members of the family (see boxed areas of FIG.2A-2B). In addition, the signature for the PDGF/VEGF family,PXCVXXXRCXGCCN, is (SEQ ID NO: 6) conserved in VEGF-2 (see FIG. 2A-2B).

[0036] The VEGF-2 polypeptide of the present invention is meant toinclude the full-length polypeptide and polynucleotide sequence thatencodes for any leader sequences and for active fragments of thefull-length polypeptide. Active fragments are meant to include anyportions of the full length amino acid sequence which have less than thefull 419 amino acids of the full length amino acid sequence as shown inSEQ ID No. 2 and FIG. 2A-2B, but still contain the eight cysteineresidues shown conserved in FIG. 2A-2B and such fragments still containVEGF-2 activity.

[0037] There are at least two alternatively spliced VEGF-2 mRNAsequences present in normal tissues. The size of the two VEGF-2 mRNAsequences which correspond to the full-length and truncated versionrespectively are shown in FIG. 3, lane 5 shows two bands indicating thepresence of the alternatively spliced mRNA encoding the VEGF-2polypeptide of the present invention.

[0038] The polynucleotide of the present invention may be in the form ofRNA or in the form of DNA, which DNA includes cDNA, genomic DNA, andsynthetic DNA. The DNA may be double-stranded or single-stranded, and ifsingle stranded may be the coding strand or non-coding (anti-sense)strand. The coding sequence which encodes the mature polypeptide may beidentical to the coding sequence shown in FIG. 1A-1C or that of thedeposited clone or may be a different coding sequence which codingsequence, as a result of the redundancy or degeneracy of the geneticcode, encodes the same mature polypeptide as the DNA of FIG. 1A-1C orthe deposited cDNA.

[0039] The polynucleotide which encodes for the mature polypeptide ofFIG. 1A-1C or for the mature polypeptide encoded by the deposited cDNAmay include: only the coding sequence for the mature polypeptide; thecoding sequence for the mature polypeptide (and optionally additionalcoding sequence) and non-coding sequence, such as introns or non-codingsequence 5′ and/or 3′ of the coding sequence for the mature polypeptide.

[0040] Thus, the term “polynucleotide encoding a polypeptide”encompasses a polynucleotide that includes only coding sequence for thepolypeptide as well as a polynucleotide that includes additional codingand/or non-coding sequence.

[0041] The present invention further relates to variants of thehereinabove described polynucleotides that encode for fragments, analogsand derivatives of the polypeptide having the deduced amino acidsequence of FIG. 1A-1C or the polypeptide encoded by the cDNA of thedeposited clone. The variant of the polynucleotide may be a naturallyoccurring allelic variant of the polynucleotide or a non-naturallyoccurring variant of the polynucleotide.

[0042] Thus, the present invention includes polynucleotides encoding thesame mature polypeptide as shown in FIG. 1A-1C or the same maturepolypeptide encoded by the cDNA of the deposited clone as well asvariants of such polynucleotides which variants encode for a fragment,derivative or analog of the polypeptide of FIG. 1A-1C or the polypeptideencoded by the cDNA of the deposited clone. Such nucleotide variantsinclude deletion variants, substitution variants and addition orinsertion variants.

[0043] As hereinabove indicated, the polynucleotide may have a codingsequence which is a naturally occurring allelic variant of the codingsequence shown in FIG. 1A-1C or of the coding sequence of the depositedclone. As known in the art, an allelic variant is an alternate form of apolynucleotide sequence that may have a substitution, deletion oraddition of one or more nucleotides, which does not substantially alterthe function of the encoded polypeptide.

[0044] The polynucleotides of the present invention may also have thecoding sequence fused in frame to a marker sequence that allows forpurification of the polypeptide of the present invention. The markersequence may be a hexa-histidine tag supplied by a pQE-9 vector toprovide for purification of the mature polypeptide fused to the markerin the case of a bacterial host, or, for example, the marker sequencemay be a hemagglutinin (HA) tag when a mammalian host, e.g. COS-7 cells,is used. The HA tag corresponds to an epitope derived from the influenzahemagglutinin protein (Wilson, I., et al., Cell, 37:767 (1984)).

[0045] The term “gene” means the segment of DNA involved in producing apolypeptide chain; it includes regions preceding and following thecoding region (leader and trailer) as well as intervening sequences(introns) between individual coding segments (exons).

[0046] Fragments of the full-length gene of the present invention may beused as a hybridization probe for a cDNA library to isolate thefull-length cDNA and to isolate other cDNAs that have a high sequencesimilarity to the gene or similar biological activity. Probes of thistype preferably have at least 30 bases and may contain, for example, 50or more bases. The probe may also be used to identify a cDNA clonecorresponding to a full-length transcript and a genomic clone or clonesthat contains the complete gene including regulatory and promotorregions, exons, and introns. An example of a screen comprises isolatingthe coding region of the gene by using the known DNA sequence tosynthesize an oligonucleotide probe. Labeled oligonucleotides having asequence complementary to that of the gene of the present invention areused to screen a library of human cDNA, genomic DNA or mRNA to determinewhich members of the library the probe hybridizes to.

[0047] The present invention further relates to polynucleotides thathybridize to the hereinabove-described sequences if there is at least70%, preferably at least 90%, and more preferably at least 95% identitybetween the sequences. The present invention particularly relates topolynucleotides that hybridize under stringent conditions to thehereinabove-described polynucleotides. As herein used, the term“stringent conditions” means hybridization will occur only if there isat least 95% and preferably at least 97% identity between the sequences.The polynucleotides which hybridize to the hereinabove describedpolynucleotides in a preferred embodiment encode polypeptides whicheither retain substantially the same biological function or activity asthe mature polypeptide encoded by the cDNAs of FIG. 1A-1C (SEQ ID NO: 1)or the deposited cDNA(s).

[0048] Alternatively, the polynucleotide may have at least 20 bases,preferably 30 bases, and more preferably at least 50 bases whichhybridize to a polynucleotide of the present invention and which has anidentity thereto, as hereinabove described, and which may or may notretain activity. For example, such polynucleotides may be employed asprobes for the polynucleotide of SEQ ID NO: 1, for example, for recoveryof the polynucleotide or as a diagnostic probe or as a PCR primer.

[0049] Thus, the present invention is directed to polynucleotides havingat least a 70% identity, preferably at least 90% and more preferably atleast a 95% identity to a polynucleotide which encodes the polypeptideof SEQ ID NO: 2 as well as fragments thereof, which fragments have atleast 30 bases and preferably at least 50 bases and to polypeptidesencoded by such polynucleotides.

[0050] The deposit(s) referred to herein will be maintained under theterms of the Budapest Treaty on the International Recognition of theDeposit of Micro-organisms for purposes of Patent Procedure. Thesedeposits are provided merely as convenience to those of skill in the artand are not an admission that a deposit is required under 35 U.S.C.§112. The sequence of the polynucleotides contained in the depositedmaterials, as well as the amino acid sequence of the polypeptidesencoded thereby, are incorporated herein by reference and arecontrolling in the event of any conflict with any description ofsequences herein. A license may be required to make, use or sell thedeposited materials, and no such license is hereby granted.

[0051] The present invention further relates to polypeptides that havethe deduced amino acid sequence of FIG. 1A-1C or which has the aminoacid sequence encoded by the deposited cDNA, as well as fragments,analogs and derivatives of such polypeptide.

[0052] The terms “fragment,” “derivative” and “analog” when referring tothe polypeptide of FIG. 1A-1C or that encoded by the deposited cDNA,means a polypeptide which retains the conserved motif of VEGF proteinsas shown in FIG. 2A-2B and essentially the same biological function oractivity.

[0053] The polypeptides of the present invention may be recombinantpolypeptides, natural polypeptides or synthetic polypeptides, preferablyrecombinant polypeptides.

[0054] The fragment, derivative or analog of the polypeptide of FIG.1A-1C or that encoded by the deposited cDNA may be (i) one in which oneor more of the amino acid residues are substituted with a conserved ornon-conserved amino acid residue (preferably a conserved amino acidresidue) and such substituted amino acid residue may or may not be oneencoded by the genetic code, or (ii) one in which one or more of theamino acid residues includes a substituent group, or (iii) one in whichthe mature polypeptide is fused with another compound, such as acompound to increase the half-life of the polypeptide (for example,polyethylene glycol), or (iv) one in which the additional amino acidsare fused to the mature polypeptide or (v) one in which comprises feweramino acid residues shown in SEQ ID No. 2 and retains the conservedmotif and yet still retains activity characteristic of the VEGF familyof polypeptides. Such fragments, derivatives and analogs are deemed tobe within the scope of those skilled in the art from the teachingsherein.

[0055] The polypeptides and polynucleotides of the present invention arepreferably provided in an isolated form, and preferably are purified tohomogeneity.

[0056] The term “isolated” means that the material is removed from itsoriginal environment (e.g., the natural environment if it is naturallyoccurring). For example, a naturally occurring polynucleotide orpolypeptide present in a living animal is not isolated, but the samepolynucleotide or polypeptide, separated from some or all of thecoexisting materials in the natural system, is isolated. Suchpolynucleotides could be part of a vector and/or such polynucleotides orpolypeptides could be part of a composition, and still be isolated inthat such vector or composition is not part of its natural environment.

[0057] The polypeptides of the present invention include the polypeptideof SEQ ID NO: 2 (in particular the mature polypeptide) as well aspolypeptides which have at least 70% similarity (preferably at least 70%identity) to the polypeptide of SEQ ID NO: 2 and more preferably atleast 90% similarity (more preferably at least 95% identity) to thepolypeptide of SEQ ID NO: 2 and still more preferably at least 95%similarity (still more preferably at least 90% identity) to thepolypeptide of SEQ ID NO: 2 and also include portions of suchpolypeptides with such portion of the polypeptide generally containingat least 30 amino acids and more preferably at least 50 amino acids.

[0058] As known in the art “similarity” between two polypeptides isdetermined by comparing the amino acid sequence and its conserved aminoacid substitutes of one polypeptide to the sequence of a secondpolypeptide.

[0059] Fragments or portions of the polypeptides of the presentinvention may be employed for producing the corresponding full-lengthpolypeptide by peptide synthesis; therefore, the fragments may beemployed as intermediates for producing the full-length polypeptides.Fragments or portions of the polynucleotides of the present inventionmay be used to synthesize full-length polynucleotides of the presentinvention.

[0060] The present invention also relates to vectors, which includepolynucleotides of the present invention, host cells which aregenetically engineered with vectors of the invention and the productionof polypeptides of the invention by recombinant techniques.

[0061] Host cells are genetically engineered (transduced or transformedor transfected) with the vectors of this invention, which may be, forexample, a cloning vector or an expression vector. The vector may be,for example, in the form of a plasmid, a viral particle, a phage, etc.The engineered host cells can be cultured in conventional nutrient mediamodified as appropriate for activating promoters, selectingtransformants or amplifying the VEGF-2 genes of the present invention.The culture conditions, such as temperature, pH and the like, are thosepreviously used with the host cell selected for expression, and will beapparent to the ordinarily skilled artisan.

[0062] The polynucleotides of the present invention may be employed forproducing polypeptides by recombinant techniques. Thus, for example, thepolynucleotide may be included in any one of a variety of expressionvectors for expressing a polypeptide. Such vectors include chromosomal,nonchromosomal and synthetic DNA sequences, e.g., derivatives of SV40;bacterial plasmids; phage DNA; baculovirus; yeast plasmids; vectorsderived from combinations of plasmids and phage DNA, viral DNA such asvaccinia, adenovirus, fowl pox virus, and pseudorabies. However, anyother vector may be used as long as it is replicable and viable in thehost.

[0063] The appropriate DNA sequence may be inserted into the vector by avariety of procedures. In general, the DNA sequence is inserted into anappropriate restriction endonuclease site(s) by procedures known in theart. Such procedures and others are deemed to be within the scope ofthose skilled in the art.

[0064] The DNA sequence in the expression vector is operatively linkedto an appropriate expression control sequence(s) (promoter) to directmRNA synthesis. As representative examples of such promoters, there maybe mentioned: LTR or SV40 promoter, the E. coli. lac or trp, the phagelambda P_(L) promoter and other promoters known to control expression ofgenes in prokaryotic or eukaryotic cells or their viruses. Theexpression vector also contains a ribosome-binding site for translationinitiation and a transcription terminator. The vector may also includeappropriate sequences for amplifying expression.

[0065] In addition, the expression vectors preferably contain one ormore selectable marker genes to provide a phenotypic trait for selectionof transformed host cells such as dihydrofolate reductase or neomycinresistance for eukaryotic cell culture, or such as tetracycline orampicillin resistance in E. coli.

[0066] The vector containing the appropriate DNA sequence as hereinabovedescribed, as well as an appropriate promoter or control sequence, maybe employed to transform an appropriate host to permit the host toexpress the protein.

[0067] As representative examples of appropriate hosts, there may bementioned: bacterial cells, such as E. coli, Streptomyces, Salmonellatyphimurium; fungal cells, such as yeast; insect cells such asDrosophila S2 and Spodoptera Sf9; animal cells such as CHO, COS or Bowesmelanoma; adenoviruses; plant cells, etc. The selection of anappropriate host is deemed to be within the scope of those skilled inthe art from the teachings herein.

[0068] More particularly, the present invention also includesrecombinant constructs comprising one or more of the sequences asbroadly described above. The constructs comprise a vector, such as aplasmid or viral vector, into which a sequence of the invention has beeninserted, in a forward or reverse orientation. In a preferred aspect ofthis embodiment, the construct further comprises regulatory sequences,including, for example, a promoter, operably linked to the sequence.Large numbers of suitable vectors and promoters are known to those ofskill in the art, and are commercially available. The following vectorsare provided by way of example. Bacterial: pQE70, pQE60, pQE-9 (Qiagen),pBS, pD10, phagescript, psiX174, pBluescript SK, pBSKS, pNH8A, pNH16a,pNH18A, pNH46A (Stratagene); ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5(Pharmacia). Eukaryotic: pWLNEO, pSV2CAT, pOG44, pXT1, pSG (Stratagene)pSVK3, pBPV, pMSG, pSVL (Pharmacia). However, any other plasmid orvector may be used as long as they are replicable and viable in thehost.

[0069] Promoter regions can be selected from any desired gene using CAT(chloramphenicol transferase) vectors or other vectors with selectablemarkers. Two appropriate vectors are pKK232-8 and pCM7. Particular namedbacterial promoters include lac, lacZ, T3, T7, gpt, lambda P_(R), P_(L)and trp. Eukaryotic promoters include CMV immediate early, HSV thymidinekinase, early and late SV40, LTRs from retrovirus, and mousemetallothionein-I. Selection of the appropriate vector and promoter iswell within the level of ordinary skill in the art.

[0070] In a further embodiment, the present invention relates to hostcells containing the above-described constructs. The host cell can be ahigher eukaryotic cell, such as a mammalian cell, or a lower eukaryoticcell, such as a yeast cell, or the host cell can be a prokaryotic cell,such as a bacterial cell. Introduction of the construct into the hostcell can be effected by calcium phosphate transfection, DEAE-Dextranmediated transfection, or electroporation. (Davis, L., Dibner, M.,Battey, I., Basic Methods in Molecular Biology, (1986)).

[0071] The constructs in host cells can be used in a conventional mannerto produce the gene product encoded by the recombinant sequence.Alternatively, the polypeptides of the invention can be syntheticallyproduced by conventional peptide synthesizers.

[0072] Mature proteins can be expressed in mammalian cells, yeast,bacteria, or other cells under the control of appropriate promoters.Cell-free translation systems can also be employed to produce suchproteins using RNAs derived from the DNA constructs of the presentinvention. Appropriate cloning and expression vectors for use withprokaryotic and eukaryotic hosts are described by Sambrook, et al.,Molecular Cloning: A Laboratory Manual, Second Edition, Cold SpringHarbor, N.Y., (1989), the disclosure of which is hereby incorporated byreference.

[0073] Transcription of the DNA encoding the polypeptides of the presentinvention by higher eukaryotes is increased by inserting an enhancersequence into the vector. Enhancers are cis-acting elements of DNA,usually about from 10 to 300 bp that act on a promoter to increase itstranscription. Examples including the SV40 enhancer on the late side ofthe replication origin bp 100 to 270, a cytomegalovirus early promoterenhancer, the polyoma enhancer on the late side of the replicationorigin, and adenovirus enhancers.

[0074] Generally, recombinant expression vectors will include origins ofreplication and selectable markers permitting transformation of the hostcell, e.g., the ampicillin resistance gene of E. coli and S. cerevisiaeTRP1 gene, and a promoter derived from a highly-expressed gene to directtranscription of a downstream structural sequence. Such promoters can bederived from operons encoding glycolytic enzymes such as3-phosphoglycerate kinase (PGK), α-factor, acid phosphatase, or heatshock proteins, among others. The heterologous structural sequence isassembled in appropriate phase with translation initiation andtermination sequences, and preferably, a leader sequence capable ofdirecting secretion of translated protein into the periplasmic space orextracellular medium. Optionally, the heterologous sequence can encode afusion protein including an N-terminal identification peptide impartingdesired characteristics, e.g., stabilization or simplified purificationof expressed recombinant product.

[0075] Useful expression vectors for bacterial use are constructed byinserting a structural DNA sequence encoding a desired protein togetherwith suitable translation initiation and termination signals in operablereading phase with a functional promoter. The vector will comprise oneor more phenotypic selectable markers and an origin of replication toensure maintenance of the vector and to, if desirable, provideamplification within the host. Suitable prokaryotic hosts fortransformation include E. coli, Bacillus subtilis, Salmonellatyphimurium and various species within the genera Pseudomonas,Streptomyces, and Staphylococcus, although others may also be employedas a matter of choice.

[0076] As a representative but nonlimiting example, useful expressionvectors for bacterial use can comprise a selectable marker and bacterialorigin of replication derived from commercially available plasmidscomprising genetic elements of the well known cloning vector pBR322(ATCC 37017). Such commercial vectors include, for example, pKK223-3(Pharmacia Fine Chemicals, Uppsala, Sweden) and GEM1 (Promega Biotec,Madison, Wis., USA). These pBR322 “backbone” sections are combined withan appropriate promoter and the structural sequence to be expressed.

[0077] Following transformation of a suitable host strain and growth ofthe host strain to an appropriate cell density, the selected promoter isinduced by appropriate means (e.g., temperature shift or chemicalinduction) and cells are cultured for an additional period.

[0078] Cells are typically harvested by centrifugation, disrupted byphysical or chemical means, and the resulting crude extract retained forfurther purification.

[0079] Microbial cells employed in expression of proteins can bedisrupted by any convenient method, including freeze-thaw cycling,sonication, mechanical disruption, or use of cell lysing agents, suchmethods are well know to those skilled in the art.

[0080] Various mammalian cell culture systems can also be employed toexpress recombinant protein. Examples of mammalian expression systemsinclude the COS-7 lines of monkey kidney fibroblasts, described byGluzman, Cell, 23:175 (1981), and other cell lines capable of expressinga compatible vector, for example, the C127, 3T3, CHO, HeLa and BHK celllines. Mammalian expression vectors will comprise an origin ofreplication, a suitable promoter and enhancer, and also any necessaryribosome binding sites, polyadenylation site, splice donor and acceptorsites, transcriptional termnination sequences, and 5′ flankingnontranscribed sequences. DNA sequences derived from the SV40 splice,and polyadenylation sites may be used to provide the requirednontranscribed genetic elements.

[0081] The polypeptides can be recovered and purified from recombinantcell cultures by methods including ammonium sulfate or ethanolprecipitation, acid extraction, anion or cation exchange chromatography,phosphocellulose chromatography, hydrophobic interaction chromatography,affinity chromatography, hydroxylapatite chromatography and lectinchromatography. Protein refolding steps can be used, as necessary, incompleting configuration of the mature protein. Finally, highperformance liquid chromatography (HPLC) can be employed for finalpurification steps.

[0082] The polypeptides of the present invention may be a naturallypurified product, or a product of chemical synthetic procedures, orproduced by recombinant techniques from a prokaryotic or eukaryotic host(for example, by bacterial, yeast, higher plant, insect and mammaliancells in culture). Depending upon the host employed in a recombinantproduction procedure, the polypeptides of the present invention may beglycosylated or may be non-glycosylated. Polypeptides of the inventionmay also include an initial methionine amino acid residue.

[0083] As shown in FIG. 8 and FIG. 9, the VEGF-2 polypeptide of SEQ IDNo. 2, minus the initial 46 amino acids, is a potent mitogen forvascular endothelial cells and stimulates their growth andproliferation. The results of a Northern blot analysis performed for theVEGF-2 nucleic acid sequence encoding this polypeptide wherein 20 ug ofRNA from several human tissues were probed with ³²P-VEGF-2, illustratesthat this protein is actively expressed in the heart and lung which isfurther evidence of mitogenic activity.

[0084] Accordingly, VEGF-2 may be employed to promote angiogenesis, forexample, to stimulate the growth of transplanted tissue where coronarybypass surgery is performed. VEGF-2 may also be employed to promotewound healing, particularly to re-vascularize damaged tissues orstimulate collateral blood flow during ischemia and where new capillaryangiogenesis is desired. VEGF-2 may be employed to treat full-thicknesswounds such as dermal ulcers, including pressure sores, venous ulcers,and diabetic ulcers. In addition, VEGF-2 may be employed to treatfull-thickness bums and injuries where a skin graft or flap is used torepair such bums and injuries. VEGF-2 may also be employed for use inplastic surgery, for example, for the repair of lacerations from traumaand cuts in association with surgery.

[0085] Along these same lines, VEGF-2 may be employed to induce thegrowth of damaged bone, periodontium or ligament tissue. VEGF-2 may alsobe employed for regenerating supporting tissues of the teeth, includingcementum and periodontal ligament, that have been damaged by disease andtrauma.

[0086] Since angiogenesis is important in keeping wounds clean andnon-infected, VEGF-2 may be employed in association with surgery andfollowing the repair of cuts. It may also be employed for the treatmentof abdominal wounds where there is a high risk of infection.

[0087] VEGF-2 may be employed for the promotion of endothelialization invascular graft surgery. In the case of vascular grafts using eithertransplanted or synthetic material, VEGF-2 can be applied to the surfaceof the graft or at the junction to promote the growth of vascularendothelial cells. VEGF-2 may also be employed to repair damage ofmyocardial tissue as a result of myocardial infarction. VEGF-2 may alsobe employed to repair the cardiac vascular system after ischemia. VEGF-2may also be employed to treat damaged vascular tissue as a result ofcoronary artery disease and peripheral and CNS vascular disease.

[0088] VEGF-2 may also be employed to coat artificial prostheses ornatural organs which are to be transplanted in the body to minimizerejection of the transplanted material and to stimulate vascularizationof the transplanted materials.

[0089] VEGF-2 may also be employed for vascular tissue repair, forexample, that occurring during arteriosclerosis and required followingballoon angioplasty where vascular tissues are damaged.

[0090] VEGF-2 nucleic acid sequences and VEGF-2 polypeptides may also beemployed for in vitro purposes related to scientific research, synthesisof DNA and manufacture of DNA vectors, and for the production ofdiagnostics and therapeutics to treat human disease. For example, VEGF-2may be employed for in vitro culturing of vascular endothelial cells,where it is added to the conditional medium in a concentration from 10pg/ml to 10 ng/ml.

[0091] Fragments of the full length VEGF-2 gene may be used as ahybridization probe for a cDNA library to isolate other genes which havea high sequence similarity to the gene or similar biological activity.Probes of this type generally have at least 50 base pairs, although theymay have a greater number of bases. The probe may also be used toidentify a cDNA clone corresponding to a full length transcript and agenomic clone or clones that contain the complete VEGF-2 gene includingregulatory and promotor regions, exons, and introns. An example of ascreen comprises isolating the coding region of the VEGF-2 gene by usingthe known DNA sequence to synthesize an oligonucleotide probe. Labeledoligonucleotides having a sequence complementary to that of the gene ofthe present invention are used to screen a library of human cDNA,genomic DNA or mRNA to determine which members of the library the probehybridizes to.

[0092] This invention provides methods for identification of VEGF-2receptors. The gene encoding the receptor can be identified by numerousmethods known to those of skill in the art, for example, ligand panningand FACS sorting (Coligan, et al., Current Protocols in Immun., 1(2),Chapter 5, (1991)). Preferably, expression cloning is employed whereinpolyadenylated RNA is prepared from a cell responsive to VEGF-2, and acDNA library created from this RNA is divided into pools and used totransfect COS cells or other cells that are not responsive to VEGF-2.Transfected cells which are grown on glass slides are exposed to labeledVEGF-2. VEGF-2 can be labeled by a variety of means including iodinationor inclusion of a recognition site for a site-specific protein kinase.Following fixation and incubation, the slides are subjected toautoradiographic analysis. Positive pools are identified and sub-poolsare prepared and retransfected using an iterative sub-pooling andrescreening process, eventually yielding a single clone that encodes theputative receptor.

[0093] As an alternative approach for receptor identification, labeledVEGF-2 can be photoaffinity linked with cell membrane or extractpreparations that express the receptor molecule. Cross-linked materialis resolved by PAGE and exposed to X-ray film. The labeled complexcontaining VEGF-2 is then excised, resolved into peptide fragments, andsubjected to protein microsequencing. The amino acid sequence obtainedfrom microsequencing would be used to design a set of degenerateoligonucleotide probes to screen a cDNA library to identify the geneencoding the putative receptor.

[0094] This invention is also related to a method of screening compoundsto identify those which are VEGF-2 agonists or antagonists. An exampleof such a method takes advantage of the ability of VEGF-2 tosignificantly stimulate the proliferation of human endothelial cells inthe presence of the comitogen Con A. Endothelial cells are obtained andcultured in 96-well flat-bottomed culture plates (Costar, Cambridge,Mass.) in a reaction mixture supplemented with Con-A (Calbiochem, LaJolla, Calif.). Con-A, polypeptides of the present invention and thecompound to be screened are added. After incubation at 37° C., culturesare pulsed with 1 Ci of ³[H]thymidine (5 Ci/mmol; 1 Ci=37 BGq; NEN) fora sufficient time to incorporate the ³[H] and harvested onto glass fiberfilters (Cambridge Technology, Watertown, Mass.). Mean ³[H]-thymidineincorporation (cpm) of triplicate cultures is determined using a liquidscintillation counter (Beckman Instruments, Irvine, Calif.). Significant³[H]thymidine incorporation, as compared to a control assay where thecompound is excluded, indicates stimulation of endothelial cellproliferation.

[0095] To assay for antagonists, the assay described above is performedand the ability of the compound to inhibit ³[H]thymidine incorporationin the presence of VEGF-2 indicates that the compound is an antagonistto VEGF-2. Alternatively, VEGF-2 antagonists may be detected bycombining VEGF-2 and a potential antagonist with membrane-bound VEGF-2receptors or recombinant receptors under appropriate conditions for acompetitive inhibition assay. VEGF-2 can be labeled, such as byradioactivity, such that the number of VEGF-2 molecules bound to thereceptor can determine the effectiveness of the potential antagonist.

[0096] Alternatively, the response of a known second messenger systemfollowing interaction of VEGF-2 and receptor would be measured andcompared in the presence or absence of the compound. Such secondmessenger systems include but are not limited to, cAMP guanylatecyclase, ion channels or phosphoinositide hydrolysis. In another method,a mammalian cell or membrane preparation expressing the VEGF-2 receptoris incubated with labeled VEGF-2 in the presence of the compound. Theability of the compound to enhance or block this interaction could thenbe measured.

[0097] Potential VEGF-2 antagonists include an antibody, or in somecases, an oligonucleotide, which bind to the polypeptide and effectivelyeliminate VEGF-2 function. Alternatively, a potential antagonist may bea closely related protein which binds to VEGF-2 receptors, however, theyare inactive forms of the polypeptide and thereby prevent the action ofVEGF-2. Examples of these antagonists include a negative dominant mutantof the VEGF-2 polypeptide, for example, one chain of the hetero-dimericform of VEGF-2 may be dominant and may be mutated such that biologicalactivity is not retained. An example of a negative dominant mutantincludes truncated versions of a dimeric VEGF-2 which is capable ofinteracting with another dimer to form wild type VEGF-2, however, theresulting homo-dimer is inactive and fails to exhibit characteristicVEGF activity.

[0098] Another potential VEGF-2 antagonist is an antisense constructprepared using antisense technology. Antisense technology can be used tocontrol gene expression through triple-helix formation or antisense DNAor RNA, both of which methods are based on binding of a polynucleotideto DNA or RNA. For example, the 5′ coding portion of the polynucleotidesequence, which encodes for the mature polypeptides of the presentinvention, is used to design an antisense RNA oligonucleotide of fromabout 10 to 40 base pairs in length. A DNA oligonucleotide is designedto be complementary to a region of the gene involved in transcription(triple helix —see Lee et al., Nucl. Acids Res., 6:3073 (1979); Cooneyet al, Science, 241:456 (1988); and Dervan et al., Science, 251: 1360(1991)), thereby preventing transcription and the production of VEGF-2.The antisense RNA oligonucleotide hybridizes to the mRNA in vivo andblocks translation of the mRNA molecule into the VEGF-2 polypeptide(Antisense−Okano, J. Neurochem., 56:560 (1991); Oligodeoxynucleotides asAntisense Inhibitors of Gene Expression, CRC Press, Boca Raton, Fla.(1988)). The oligonucleotides described above can also be delivered tocells such that the antisense RNA or DNA may be expressed in vivo toinhibit production of VEGF-2.

[0099] Potential VEGF-2 antagonists also include small molecules whichbind to and occupy the active site of the polypeptide thereby making thecatalytic site inaccessible to substrate such that normal biologicalactivity is prevented. Examples of small molecules include but are notlimited to small peptides or peptide-like molecules.

[0100] The antagonists may be employed to treat limit angiogenesisnecessary for solid tumor metastasis.

[0101] The mRNA encoding for VEGF-2 is found to be expressed at moderatelevels in at least two breast tumor cell lines which is indicative ofthe role of VEGF-2 polypeptides in the malignant phenotype. Gliomas arealso a type of neoplasia which may be treated with the antagonists ofthe present invention.

[0102] The antagonists may also be used to treat chronic inflammationcaused by increased vascular permeability. In addition to thesedisorders, the antagonists may also be employed to treat retinopathyassociated with diabetes, rheumatoid arthritis and psoriasis.

[0103] The antagonists may be employed in a composition with apharmaceutically acceptable carrier, e.g., as hereinafter described.

[0104] The VEGF-2 polypeptides and agonists and antagonists may beemployed in combination with a suitable pharmaceutical carrier. Suchcompositions comprise a therapeutically effective amount of thepolypeptide or agonist or antagonist, and a pharmaceutically acceptablecarrier or excipient. Such a carrier includes but is not limited tosaline, buffered saline, dextrose, water, glycerol, ethanol, andcombinations thereof. The formulation should suit the mode ofadministration.

[0105] The invention also provides a pharmaceutical pack or kitcomprising one or more containers filled with one or more of theingredients of the pharmaceutical compositions of the invention.Associated with such container(s) can be a notice in the form prescribedby a governmental agency regulating the manufacture, use or sale ofpharmaceuticals or biological products, which notice reflects approvalby the agency of manufacture, use or sale for human administration. Inaddition, the pharmaceutical compositions may be employed in conjunctionwith other therapeutic compounds.

[0106] The pharmaceutical compositions may be administered in aconvenient manner such as by the topical, intravenous, intraperitoneal,intramuscular, intratumor, subcutaneous, intranasal or intradermalroutes. The pharmaceutical compositions are administered in an amountwhich is effective for treating and/or prophylaxis of the specificindication. In general, the pharmaceutical compositions are administeredin an amount of at least about 10 ug/kg body weight and in most casesthey will be administered in an amount not in excess of about 8 mg/Kgbody weight per day. In most cases, the dosage is from about 10 ug/kg toabout 1 mg/kg body weight daily, taking into account the routes ofadministration, symptoms, etc.

[0107] The VEGF-2 polypeptides, and agonists or antagonists which arepolypeptides may also be employed in accordance with the presentinvention by expression of such polypeptide in vivo, which is oftenreferred to as “gene therapy.”

[0108] Thus, for example, cells such as bone marrow cells may beengineered with a polynucleotide (DNA or RNA) encoding for thepolypeptide ex vivo, the engineered cells are then provided to a patientto be treated with the polypeptide. Such methods are well-known in theart. For example, cells may be engineered by procedures known in the artby use of a retroviral particle containing RNA encoding the polypeptideof the present invention.

[0109] Similarly, cells may be engineered in vivo for expression of apolypeptide in vivo, for example, by procedures known in the art. Asknown in the art, a producer cell for producing a retroviral particlecontaining RNA encoding a polypeptide of the present invention may beadministered to a patient for engineering cells in vivo and expressionof the polypeptide in vivo. These and other methods for administering apolypeptide of the present invention by such methods should be apparentto those skilled in the art from the teachings of the present invention.For example, the expression vehicle for engineering cells may be otherthan a retroviral particle, for example, an adenovirus, which may beused to engineer cells in vivo after combination with a suitabledelivery vehicle.

[0110] Retroviruses from which the retroviral plasmid vectorshereinabove mentioned may be derived include, but are not limited to,Moloney Murine Leukemia Virus, spleen necrosis virus, retroviruses suchas Rous Sarcoma Virus, Harvey Sarcoma Virus, avian leukosis virus,gibbon ape leukemia virus, human immunodeficiency virus, adenovirus,Myeloproliferative Sarcoma Virus, and mammary tumor virus. In oneembodiment, the retroviral plasmid vector is derived from Moloney MurineLeukemia Virus.

[0111] The vector includes one or more promoters. Suitable promoterswhich may be employed include, but are not limited to, the retroviralLTR; the SV40 promoter; and the human cytomegalovirus (CMV) promoterdescribed in Miller, et al., Biotechniques, Vol. 7, No. 9, 980-990(1989), or any other promoter (e.g., cellular promoters such aseukaryotic cellular promoters including, but not limited to, thehistone, pol III, and β-actin promoters). Other viral promoters whichmay be employed include, but are not limited to, adenovirus promoters,thymidine kinase (TK) promoters, and B19 parvovirus promoters. Theselection of a suitable promoter will be apparent to those skilled inthe art from the teachings contained herein.

[0112] The nucleic acid sequence encoding the polypeptide of the presentinvention is under the control of a suitable promoter. Suitablepromoters which may be employed include, but are not limited to,adenoviral promoters, such as the adenoviral major late promoter; orhetorologous promoters, such as the cytomegalovirus (CMV) promoter; therespiratory syncytial virus (RSV) promoter; inducible promoters, such asthe MMT promoter, the metallothionein promoter; heat shock promoters;the albumin promoter; the ApoAI promoter; human globin promoters; viralthymidine kinase promoters, such as the Herpes Simplex thymidine kinasepromoter; retroviral LTRs (including the modified retroviral LTRshereinabove described); the β-actin promoter; and human growth hormonepromoters. The promoter also may be the native promoter which controlsthe gene encoding the polypeptide.

[0113] The retroviral plasmid vector is employed to transduce packagingcell lines to form producer cell lines. Examples of packaging cellswhich may be transfected include, but are not limited to, the PE501,PA317, ψ-2, ψ-AM, PA12, T19-14X, VT-19-17-H2, ψCRE, ψCRIP, GP+E-86,GP+envAm12, and DAN cell lines as described in Miller, Human GeneTherapy, Vol. 1, pgs. 5-14 (1990), which is incorporated herein byreference in its entirety. The vector may transduce the packaging cellsthrough any means known in the art. Such means include, but are notlimited to, electroporation, the use of liposomes, and CaPO₄precipitation. In one alternative, the retroviral plasmid vector may beencapsulated into a liposome, or coupled to a lipid, and thenadministered to a host.

[0114] The producer cell line generates infectious retroviral vectorparticles which include the nucleic acid sequence(s) encoding thepolypeptides. Such retroviral vector particles then may be employed, totransduce eukaryotic cells, either in vitro or in vivo. The transducedeukaryotic cells will express the nucleic acid sequence(s) encoding thepolypeptide. Eukaryotic cells which may be transduced include, but arenot limited to, embryonic stem cells, embryonic carcinoma cells, as wellas hematopoietic stem cells, hepatocytes, fibroblasts, myoblasts,keratinocytes, endothelial cells, and bronchial epithelial cells.

[0115] This invention is also related to the use of the VEGF-2 gene aspart of a diagnostic assay for detecting diseases or susceptibility todiseases related to the presence of mutations in VEGF-2 nucleic acidsequences.

[0116] Individuals carrying mutations in the VEGF-2 gene may be detectedat the DNA level by a variety of techniques. Nucleic acids for diagnosismay be obtained from a patient's cells, such as from blood, urine,saliva, tissue biopsy and autopsy material. The genomic DNA may be useddirectly for detection or may be amplified enzymatically by using PCR(Saiki et al., Nature, 324:163-166 (1986)) prior to analysis. RNA orcDNA may also be used for the same purpose. As an example, PCR primerscomplementary to the nucleic acid encoding VEGF-2 can be used toidentify and analyze VEGF-2 mutations. For example, deletions andinsertions can be detected by a change in size of the amplified productin comparison to the normal genotype. Point mutations can be identifiedby hybridizing amplified DNA to radiolabeled VEGF-2 RNA oralternatively, radiolabeled VEGF-2 antisense DNA sequences. Perfectlymatched sequences can be distinguished from mismatched duplexes by RNaseA digestion or by differences in melting temperatures.

[0117] Genetic testing based on DNA sequence differences may be achievedby detection of alteration in electrophoretic mobility of DNA fragmentsin gels with or without denaturing agents. Small sequence deletions andinsertions can be visualized by high resolution gel electrophoresis. DNAfragments of different sequences may be distinguished on denaturingformamide gradient gels in which the mobilities of different DNAfragments are retarded in the gel at different positions according totheir specific melting or partial melting temperatures (see, e.g., Myerset al., Science, 230:1242 (1985)).

[0118] Sequence changes at specific locations may also be revealed bynuclease protection assays, such as RNase and S1 protection or thechemical cleavage method (e.g., Cotton et al., PNAS, USA, 85:4397-4401(1985)).

[0119] Thus, the detection of a specific DNA sequence may be achieved bymethods such as hybridization, RNase protection, chemical cleavage,direct DNA sequencing or the use of restriction enzymes, (e.g.,Restriction Fragment Length Polymorphisms (RFLP)) and Southern blottingof genomic DNA.

[0120] In addition to more conventional gel-electrophoresis and DNAsequencing, mutations can also be detected by in situ analysis.

[0121] The present invention also relates to a diagnostic assay fordetecting altered levels of VEGF-2 protein in various tissues since anover-expression of the proteins compared to normal control tissuesamples may detect the presence of a disease or susceptibility to adisease, for example, abnormal cellular differentiation. Assays used todetect levels of VEGF-2 protein in a sample derived from a host arewell-known to those of skill in the art and include radioimmunoassays,competitive-binding assays, Western Blot analysis, ELISA assays and“sandwich” assay. An ELISA assay (Coligan, et al., Current Protocols inImmunology, 1(2), Chapter 6, (1991)) initially comprises preparing anantibody specific to the VEGF-2 antigen, preferably a monoclonalantibody. In addition a reporter antibody is prepared against themonoclonal antibody. To the reporter antibody is attached a detectablereagent such as radioactivity, fluorescence or, in this example, ahorseradish peroxidase enzyme. A sample is removed from a host andincubated on a solid support, e.g. a polystyrene dish, that binds theproteins in the sample. Any free protein binding sites on the dish arethen covered by incubating with a non-specific protein, such as, bovineserum albumen. Next, the monoclonal antibody is incubated in the dishduring which time the monoclonal antibodies attach to any VEGF-2proteins attached to the polystyrene dish. All unbound monoclonalantibody is washed out with buffer. The reporter antibody linked tohorseradish peroxidase is placed in the dish resulting in binding of thereporter antibody to any monoclonal antibody bound to VEGF-2. Unattachedreporter antibody is then washed out. Peroxidase substrates are thenadded to the dish and the amount of color developed in a given timeperiod is a measurement of the amount of VEGF-2 protein present in agiven volume of patient sample when compared against a standard curve.

[0122] A competition assay may be employed wherein antibodies specificto VEGF-2 are attached to a solid support. Polypeptides of the presentinvention are then labeled, for example, by radioactivity, and a samplederived from the host are passed over the solid support and the amountof label detected, for example by liquid scintillation chromatography,can be correlated to a quantity of VEGF-2 in the sample.

[0123] A “sandwich” assay is similar to an ELISA assay. In a “sandwich”assay VEGF-2 is passed over a solid support and binds to antibodyattached to a solid support. A second antibody is then bound to theVEGF-2. A third antibody which is labeled and specific to the secondantibody is then passed over the solid support and binds to the secondantibody and an amount can then be quantified.

[0124] The sequences of the present invention are also valuable forchromosome identification. The sequence is specifically targeted to andcan hybridize with a particular location on an individual humanchromosome. Moreover, there is a current need for identifying particularsites on the chromosome. Few chromosome marking reagents based on actualsequence data (repeat polymorphism's) are presently available formarking chromosomal location. The mapping of DNAs to chromosomesaccording to the present invention is an important first step incorrelating those sequences with genes associated with disease.

[0125] Briefly, sequences can be mapped to chromosomes by preparing PCRprimers (preferably 15-25 bp) from the cDNA. Computer analysis of thecDNA is used to rapidly select primers that do not span more than oneexon in the genomic DNA, thus complicating the amplification process.These primers are then used for PCR screening of somatic cell hybridscontaining individual human chromosomes. Only those hybrids containingthe human gene corresponding to the primer will yield an amplifiedfragment.

[0126] PCR mapping of somatic cell hybrids is a rapid procedure forassigning a particular DNA to a particular chromosome. Using the presentinvention with the same oligonucleotide primers, sublocalization can beachieved with panels of fragments from specific chromosomes or pools oflarge genomic clones in an analogous manner. Other mapping strategiesthat can similarly be used to map to its chromosome include in situhybridization, prescreening with labeled flow-sorted chromosomes andpreselection by hybridization to construct chromosome specific-cDNAlibraries.

[0127] Fluorescence in situ hybridization (FISH) of a cDNA clone to ametaphase chromosomal spread can be used to provide a precisechromosomal location in one step. This technique can be used with cDNAas short as 50 or 60 bases. For a review of this technique, see Verma etal., Human Chromosomes: a Manual of Basic Techniques. Pergamon Press,New York (1988).

[0128] Once a sequence has been mapped to a precise chromosomallocation, the physical position of the sequence on the chromosome can becorrelated with genetic map data. (Such data are found, for example, inV. McKusick, Mendelian Inheritance in Man (available on line throughJohns Hopkins University Welch Medical Library). The relationshipbetween genes and diseases that have been mapped to the same chromosomalregion are then identified through linkage analysis (coinheritance ofphysically adjacent genes).

[0129] Next, it is necessary to determine the differences in the cDNA orgenomic sequence between affected and unaffected individuals. If amutation is observed in some or all of the affected individuals but notin any normal individuals, then the mutation is likely to be thecausative agent of the disease.

[0130] With current resolution of physical mapping and genetic mappingtechniques, a cDNA precisely localized to a chromosomal regionassociated with the disease could be one of between 50 and 500 potentialcausative genes. (This assumes 1 megabase mapping resolution and onegene per 20 kb).

[0131] The polypeptides, their fragments or other derivatives, oranalogs thereof, or cells expressing them can be used as an immunogen toproduce antibodies thereto. These antibodies can be, for example,polyclonal or monoclonal antibodies. The present invention also includeschimeric, single chain, and humanized antibodies, as well as Fabfragments, or the product of an Fab expression library. Variousprocedures known in the art may be used for the production of suchantibodies and fragments.

[0132] Antibodies generated against the polypeptide corresponding to asequence of the present invention can be obtained by direct injection ofthe polypeptide into an animal or by administering the polypeptide to ananimal, preferably a nonhuman. The antibody so obtained will then bindthe polypeptide itself. In this manner, even a sequence encoding only afragment of the polypeptide can be used to generate antibodies bindingthe whole native polypeptide. Such antibodies can then be used toisolate the polypeptide from tissue expressing that polypeptide. Forpreparation of monoclonal antibodies, any technique which providesantibodies produced by continuous cell line cultures can be used.Examples include the hybridoma technique (Kohler and Milstein, 1975,Nature, 256:495-497), the trioma technique, the human B-cell hybridomatechnique (Kozbor et al., 1983, Immunology Today 4:72), and theEBV-hybridoma technique to produce human monoclonal antibodies (Cole, etal., 1985, in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss,Inc., pp. 77-96).

[0133] Techniques described for the production of single chainantibodies (U.S. Pat. No. 4,946,778) can be adapted to produce singlechain antibodies to immunogenic polypeptide products of this invention.Also, transgenic mice may be used to express humanized antibodies toimmunogenic polypeptide products of this invention.

[0134] The present invention will be further described with reference tothe following examples; however, it is to be understood that the presentinvention is not limited to such examples. All parts or amounts, unlessotherwise specified, are by weight.

[0135] In order to facilitate understanding of the following examples,certain frequently occurring methods and/or terms will be described.

[0136] “Plasmids” are designated by a lower case “p” preceded and/orfollowed by capital letters and/or numbers. The starting plasmids hereinare either commercially available, publicly available on an unrestrictedbasis, or can be constructed from available plasmids in accord withpublished procedures. In addition, equivalent plasmids to thosedescribed are known in the art and will be apparent to the ordinarilyskilled artisan.

[0137] “Digestion” of DNA refers to catalytic cleavage of the DNA with arestriction enzyme that acts only at certain sequences in the DNA. Thevarious restriction enzymes used herein are commercially available andtheir reaction conditions, cofactors and other requirements were used aswould be known to the ordinarily skilled artisan. For analyticalpurposes, typically 1 ug of plasmid or DNA fragment is used with about 2units of enzyme in about 20 ul of buffer solution. For the purpose ofisolating DNA fragments for plasmid construction, typically 5 to 50 ugof DNA are digested with 20 to 250 units of enzyme in a larger volume.Appropriate buffers and substrate amounts for particular restrictionenzymes are specified by the manufacturer. Incubation times of about 1hour at 37° C. are ordinarily used, but may vary in accordance with thesupplier's instructions. After digestion the reaction is electrophoreseddirectly on a polyacrylamide gel to isolate the desired fragment.

[0138] Size separation of the cleaved fragments is performed using 8percent polyacrylamide gel described by Goeddel, D. et al., NucleicAcids Res., 8:4057 (1980).

[0139] “Oligonucleotides” refers to either a single strandedpolydeoxynucleotide or two complementary polydeoxynucleotide strandswhich may be chemically synthesized. Such synthetic oligonucleotideshave no 5′ phosphate and thus will not ligate to another oligonucleotidewithout adding a phosphate with an ATP in the presence of a kinase. Asynthetic oligonucleotide will ligate to a fragment that has not beendephosphorylated.

[0140] “Ligation” refers to the process of forming phosphodiester bondsbetween two double stranded nucleic acid fragments (Maniatis, T., etal., Id., p. 146). Unless otherwise provided, ligation may beaccomplished using known buffers and conditions with 10 units of T4 DNAligase (“ligase”) per 0.5 ug of approximately equimolar amounts of theDNA fragments to be ligated.

[0141] Unless otherwise stated, transformation was performed asdescribed by the method of Graham, F. and Van der Eb, A., Virology,52:456-457 (1973).

[0142] EXAMPLE 1

Expression Pattern of VEGF-2 in Human Tissues and Breast Cancer CellLines

[0143] Northern blot analysis was carried out to examine the levels ofexpression of the VEGF-2 gene in human tissues and human breast cancercell lines. Total cellular RNA samples were isolated with RNAzol™ Bsystem (Biotecx Laboratories, Inc.). About 10 ug of total RNA isolatedfrom each breast tissue and cell line specified was separated on 1%agarose gel and blotted onto a nylon filter, (Molecular Cloning,Sambrook Fritsch, and Maniatis, Cold Spring Harbor Press, 1989). Thelabeling reaction was done according to the Stratagene Cloning Systems,Inc., Prime-It kit with 50 ng DNA fragment. The labeled DNA was purifiedwith a Select-G-50 column from 5 Prime—3 Prime, Inc, Boulder, Colo.,USA. The filter was then hybridized with radioactively labeled fulllength VEGF-2 gene at 1,000,000 cpm/ml in 0.5 M NaPO₄ and 7% SDSovernight at 65° C. After washing twice at room temperature and twice at60° C. with 0.5×SSC, 0.1% SDS, the filters were then exposed at −70° C.overnight with an intensifying screen. A message of 1.6 Kb was observedin 2 breast cancer cell lines.

EXAMPLE 2 Cloning and Expression of VEGF-2 Using the BaculovirusExpression System

[0144] The DNA sequence encoding the VEGF-2 protein without 46 aminoacids at the N-terminus, ATCC # 97149, was amplified using PCRoligonucleotide primers corresponding to the 5′ and 3′ sequences of thegene:

[0145] The 5′ primer has the sequence TGT AAT ACG ACT CAC TAT AGG GATCCC GCC ATG GAG GCC ACG GCT TAT GC (SEQ ID NO: 7) and contains a BamH1restriction enzyme site (in bold) and 17 nucleotide nucleotide sequencecomplementary to the 5′ sequence of VEGF-2 (nt. 150-166).

[0146] The 3′ primer has the sequence GATC TCT AGA TTA GCT CAT TTG TGGTCT (SEQ ID NO: 8) and contains the cleavage site for the restrictionenzyme XbaI and 18 nucleotides complementary to the 3′ sequence ofVEGF-2, including the stop codon and 15 nt sequence before stop codon.

[0147] The amplified sequences were isolated from a 1% agarose gel usinga commercially available kit (“Geneclean,” BIO 101, Inc., La Jolla,Calif.). The fragment was then digested with the endonuclease BamH1 andXbaI and then purified again on a 1% agarose gel. This fragment wasligated to pAcGP67A baculovirus transfer vector (PHarmingen) at theBamH1 and XbaI sites. Through this ligation, VEGF-2 cDNA was cloned inframe with the signal sequence of baculovirus gp67 gene and was locatedat the 3′ end of the signal sequence in the vector. This is designatedpAcGP67A-VEGF-2.

[0148] To clone VEGF-2 with the signal sequence of gp67 gene to the pRG1vector for expression, VEGF-2 with the signal sequence and some upstreamsequence were excised from the pAcGP67A-VEGF-2 plasmid at the Xhorestriction endonuclease site located upstream of the VEGF-2 cDNA and atthe XbaI restriction endonuclease site by XhoI and XbaI restrictionenzyme. This fragment was separated from the rest of vector on a 1%agarose gel and was purified using “Geneclean” kit. It was designatedF2.

[0149] The PRG1 vector (modification of pVL941 vector) is used for theexpression of the VEGF-2 protein using the baculovirus expression system(for review see: Summers, M. D. and Smith, G. E. 1987, A manual ofmethods for baculovirus vectors and insect cell culture procedures,Texas Agricultural Experimental Station Bulletin No. 1555). Thisexpression vector contains the strong polyhedrin promoter of theAutographa californica nuclear polyhedrosis virus (AcMNPV) followed bythe recognition sites for the restriction endonucleases BamH1, Sma1,XbaI, BglII and Asp718. A site for restriction endonuclease Xho1 islocated upstream of Bam-H1 site. The sequence between Xho1 and BamHI isthe same as that in PAcGp67A (static on tape) vector. Thepolyadenylation site of the simian virus (SV)40 is used for efficientpolyadenylation. For an easy selection of recombinant virus thebeta-galactosidase gene from E.coli is inserted in the same orientationas the polyhedrin promoter followed by the polyadenylation signal of thepolyhedrin gene. The polyhedrin sequences are flanked at both sides byviral sequences for the cell-mediated homologous recombination ofcotransfected wild-type viral DNA. Many other baculovirus vectors couldbe used in place of pRG1 such as pAc373, pVL941 and pAcIM1 (Luckow, V.A. and Summers, M. D., Virology, 170:31-39).

[0150] The plasmid was digested with the restriction enzymes XboI andXbaI and then dephosphorylated using calf intestinal phosphatase byprocedures known in the art. The DNA was then isolated from a 1% agarosegel using the commercially available kit (“Geneclean” BIO 101 Inc., LaJolla, Calif.). This vector DNA is designated V2.

[0151] Fragment F2 and the dephosphorylated plasmid V2 were ligated withT4 DNA ligase. E.coli HB101 cells were then transformed and bacteriaidentified that contained the plasmid (pBac gp67-VEGF-2) with the VEGF-2gene using the enzymes BamH1 and XbaI. The sequence of the clonedfragment was confirmed by DNA sequencing.

[0152] 5 μg of the plasmid pBac gp67-VEGF-2 was cotransfected with 1.0μg of a commercially available linearized baculovirus (“BaculoGold™baculovirus DNA”, Pharmingen, San Diego, Calif.) using the lipofectionmethod (Felgner et al. Proc. Natl. Acad. Sci. USA, 84:7413-7417 (1987)).

[0153]1 μg of BaculoGold™ virus DNA and 5 μg of the plasmid pBacgp67-VEGF-2 were mixed in a sterile well of a microtiter platecontaining 50 μl of serum free Grace's medium (Life Technologies Inc.,Gaithersburg, Md.). Afterwards 10 μl Lipofectin plus 90 μl Grace'smedium were added, mixed and incubated for 15 minutes at roomtemperature. Then the transfection mixture was added dropwise to the Sf9insect cells (ATCC CRL 1711) seeded in a 35 mm tissue culture plate with1 ml Grace's medium without serum. The plate was rocked back and forthto mix the newly added solution. The plate was then incubated for 5hours at 27° C. After 5 hours the transfection solution was removed fromthe plate and 1 ml of Grace's insect medium supplemented with 10% fetalcalf serum was added. The plate was put back into an incubator andcultivation continued at 27° C. for four days.

[0154] After four days the supernatant was collected and a plaque assayperformed similar as described by Summers and Smith (supra). As amodification an agarose gel with “Blue Gal” (Life Technologies Inc.,Gaithersburg) was used which allows an easy isolation of blue stainedplaques. (A detailed description of a “plaque assay” can also be foundin the user's guide for insect cell culture and baculovirologydistributed by Life Technologies Inc., Gaithersburg, page 9-10).

[0155] Four days after the serial dilution, the virus was added to thecells, blue stained plaques were picked with the tip of an Eppendorfpipette. The agar containing the recombinant viruses was thenresuspended in an Eppendorf tube containing 200 μl of Grace's medium.The agar was removed by a brief centrifugation and the supernatantcontaining the recombinant baculovirus was used to infect Sf9 cellsseeded in 35 mm dishes. Four days later the supernatants of theseculture dishes were harvested and then stored at 4° C.

[0156] Sf9 cells were grown in Grace's medium supplemented with 10%heat-inactivated FBS. The cells were infected with the recombinantbaculovirus V-gp67-VEGF-2 at a multiplicity of infection (MOI) of 1. Sixhours later the medium was removed and replaced with SF900 II mediumminus methionine and cysteine (Life Technologies Inc., Gaithersburg). 42hours later 5 μCi of ³⁵5-methionine and 5 μCi 35t cysteine (Amersham)were added. The cells were further incubated for 16 hours before theywere harvested by centrifugation and the labelled proteins visualized bySDS-PAGE and autoradiography.

[0157] Protein from the medium and cytoplasm of the Sf9 cells wasanalyzed by SDS-PAGE under reducing and non-reducing conditions. SeeFIG. 4A-4B. The medium was dialyzed against 50 mM MES, pH 5.8.Precpitates were obtained after dialysis and resuspended in 100 mMNaCitrate, pH 5.0. The resuspended precipitate was analyzed again bySDS-PAGE and was stained with Coomassie Brilliant Blue. See FIG. 5.

[0158] The medium supernatant was also diluted 1:10 in 50 mM MES, pH 5.8and applied to an SP-650M column (1.0×6.6 cm, Toyopearl) at a flow rateof 1 ml/min. Protein was eluted with step gradients at 200, 300 and 500mM NaCl. The VEGF-2 was obtained using the elution at 500 mM. The eluatewas analyzed by SDS-PAGE in the presence or absence of reducing agent,-mercaptoethanol and stained by Coommassie Brilliant Blue. See FIG. 6.

EXAMPLE 3 Expression of Recombinant VEGF-2 in COS cells

[0159] The expression of plasmid, VEGF-2-HA is derived from a vectorpcDNAI/Amp (Invitrogen) containing: 1) SV40 origin of replication, 2)ampicillin resistance gene, 3) E.coli replication origin, 4) CMVpromoter followed by a polylinker region, an SV40 intron andpolyadenylation site. A DNA fragment encoding the entire VEGF-2precursor and a HA tag fused in frame to its 3′ end was cloned into thepolylinker region of the vector, therefore, the recombinant proteinexpression is directed under the CMV promoter. The HA tag corresponds toan epitope derived from the influenza hemagglutinin protein aspreviously described (I. Wilson, H. Niman, R. Heighten, A Cherenson, M.Connolly, and R. Lerner, 1984, Cell 37:767, (1984)). The infusion of HAtag to the target protein allows easy detection of the recombinantprotein with an antibody that recognizes the HA epitope. The plasmidconstruction strategy is described as follows:

[0160] The DNA sequence encoding VEGF-2, ATCC # 97149, was constructedby PCR using two primers: the 5′ primer (CGC GGA TCC ATG ACT GTA CTC TACCCA) (SEQ ID NO: 9) contains a BamH1 site followed by 18 nucleotides ofVEGF-2 coding sequence starting from the initiation codon; the 3′sequence (CGC TCT AGA TCA AGC GTA GTC TGG GAC GTC GTA TGG GTA CTC GAGGCT CAT TTG TGG TCT 3) (SEQ ID NO: 10) contains complementary sequencesto an XbaI site, HA tag, XhoI site, and the last 15 nucleotides of theVEGF-2 coding sequence (not including the stop codon). Therefore, thePCR product contains a BamHI site, coding sequence followed by an XhoIrestriction endonuclease site and HA tag fused in frame, a translationtermination stop codon next to the HA tag, and an XbaI site. The PCRamplified DNA fragment and the vector, pcDNAI/Amp, were digested withBamH1 and XbaI restriction enzyme and ligated. The ligation mixture wastransformed into E. coli strain SURE (Stratagene Cloning Systems, LaJolla, Calif. 92037) the transformed culture was plated on ampicillinmedia plates and resistant colonies were selected. Plasmid DNA wasisolated from transformants and examined by restriction analysis for thepresence of the correct fragment. For expression of the recombinantVEGF-2, COS cells were transfected with the expression vector byDEAE-DEXTRAN method (J. Sambrook, E. Fritsch, T. Maniatis, MolecularCloning: A Laboratory Manual, Cold Spring Laboratory Press, (1989)). Theexpression of the VEGF-2-HA protein was detected by radiolabelling andimmunoprecipitation method (E. Harlow, D. Lane, Antibodies: A LaboratoryManual, Cold Spring Harbor Laboratory Press, (1988)). Cells werelabelled for 8 hours with ³⁵S-cysteine two days post transfection.Culture media was then collected and cells were lysed with detergent(RIPA buffer (150 mM NaCl, 1% NP-40, 0.1% SDS, 1% NP-40, 0.5% DOC, 50mMTris, pH 7.5) (Wilson, I. et al., Id. 37:767 (1984)). Both cell lysateand culture media were precipitated with an HA specific monoclonalantibody. Proteins precipitated were analyzed on 15% SDS-PAGE gels.

EXAMPLE 4 The Effect of Partially-Purified VEGF-2 Protein on the Growthof Vascular Endothelial Cells

[0161] On day 1, human umbilical vein endothelial cells (HUVEC) wereseeded at 2-5×10⁴ cells/35 mm dish density in M199 medium containing 4%fetal bovine serum (FBS), 16 units/ml heparin, and 50 units/mlendothelial cell growth supplements (ECGS, Biotechnique, Inc.). On day2, the medium was replaced with M199 containing 10% FBS, 8 units/mlheparin. VEGF-2 protein of SEQ ID NO. 2 minus the initial 45 amino acidresidues, (VEGF) and basic FGF (bFGF) were added, at the concentrationshown. On days 4 & 6, the medium was replaced. On day 8, cell number wasdetermined with a Coulter Counter (See FIG. 8).

EXAMPLE 5 The Effect of Purified VEGF-2 Protein on the Growth ofVascular Endothelial Cells

[0162] On day 1, human umbilical vein endothelial cells (HUVEC) wereseeded at 2-5 ×10⁴ cells/35 mm dish density in M199 medium containing 4%fetal bovine serum (FBS), 16 units/ml heparin, 50 units/ml endothelialcell growth supplements (ECGS, Biotechnique, Inc.). On day 2, the mediumwas replaced with M199 containing 10% FBS, 8 units/ml heparin. PurifiedVEGF-2 protein of SEQ ID No. 2 minus initial 45 amino acid residues wasadded to the medium at this point. On days 4 & 6, the medium wasreplaced with fresh medium and supplements. On day 8, cell number wasdetermined with a Coulter Counter (See FIG. 9).

EXAMPLE 6 Expression via Gene Therapy

[0163] Fibroblasts are obtained from a subject by skin biopsy. Theresulting tissue is placed in tissue-culture medium and separated intosmall pieces. Small chunks of the tissue are placed on a wet surface ofa tissue culture flask, approximately ten pieces are placed in eachflask. The flask is turned upside down, closed tight and left at roomtemperature over night. After 24 hours at room temperature, the flask isinverted and the chunks of tissue remain fixed to the bottom of theflask and fresh media (e.g., Ham's F12 media, with 10% FBS, penicillinand streptomycin, is added. This is then incubated at 37° C. forapproximately one week. At this time, fresh media is added andsubsequently changed every several days. After an additional two weeksin culture, a monolayer of fibroblasts emerge. The monolayer istrypsinized and scaled into larger flasks.

[0164] pMV-7 (Kirschmeier, P. T. et al, DNA, 7:219-25 (1988) flanked bythe long terminal repeats of the Moloney murine sarcoma virus, isdigested with EcoRI and HindIII and subsequently treated with calfintestinal phosphatase. The linear vector is fractionated on agarose geland purified, using glass beads.

[0165] The cDNA encoding a polypeptide of the present invention isamplified using PCR primers which correspond to the 5′ and 3′ endsequences respectively. The 5′ primer containing an EcoRI site and the3′ primer further includes a HindIII site. Equal quantities of theMoloney murine sarcoma virus linear backbone and the amplified EcoRI andHindIII fragment are added together, in the presence of T4 DNA ligase.The resulting mixture is maintained under conditions appropriate forligation of the two fragments. The ligation mixture is used to transformbacteria HB101, which are then plated onto agar-containing kanamycin forthe purpose of confirming that the vector had the gene of interestproperly inserted.

[0166] The amphotropic pA317 or GP+am12 packaging cells are grown intissue culture to confluent density in Dulbecco's Modified Eagles Medium(DMEM) with 10% calf serum (CS), penicillin and streptomycin. The MSVvector containing the gene is then added to the media and the packagingcells are transduced with the vector. The packaging cells now produceinfectious viral particles containing the gene (the packaging cells arenow referred to as producer cells).

[0167] Fresh media is added to the transduced producer cells, andsubsequently, the media is harvested from a 10 cm plate of confluentproducer cells. The spent media, containing the infectious viralparticles, is filtered through a millipore filter to remove detachedproducer cells and this media is then used to infect fibroblast cells.Media is removed from a sub-confluent plate of fibroblasts and quicklyreplaced with the media from the producer cells. This media is removedand replaced with fresh media. If the titer of virus is high, thenvirtually all fibroblasts will be infected and no selection is required.If the titer is very low, then it is necessary to use a retroviralvector that has a selectable marker, such as neo or his.

[0168] The engineered fibroblasts are then injected into the host,either alone or after having been grown to confluence on cytodex 3microcarrier beads. The fibroblasts now produce the protein product.

[0169] Numerous modifications and variations of the present inventionare possible in light of the above teachings and, therefore, within thescope of the appended claims, the invention may be practiced otherwisethan as particularly described.

1 10 1 1674 DNA Homo sapiens CDS (12)..(1268) sig_peptide (12)..(149)mat_peptide (150)..(1268) 1 gtccttccac c atg cac tcg ctg ggc ttc ttc tctgtg gcg tgt tct ctg 50 Met His Ser Leu Gly Phe Phe Ser Val Ala Cys SerLeu -45 -40 -35 ctc gcc gct gcg ctg ctc ccg ggt cct cgc gag gcg ccc gccgcc gcc 98 Leu Ala Ala Ala Leu Leu Pro Gly Pro Arg Glu Ala Pro Ala AlaAla -30 -25 -20 gcc gcc ttc gag tcc gga ctc gac ctc tcg gac gcg gag cccgac gcg 146 Ala Ala Phe Glu Ser Gly Leu Asp Leu Ser Asp Ala Glu Pro AspAla -15 -10 -5 ggc gag gcc acg gct tat gca agc aaa gat ctg gag gag cagtta cgg 194 Gly Glu Ala Thr Ala Tyr Ala Ser Lys Asp Leu Glu Glu Gln LeuArg -1 1 5 10 15 tct gtg tcc agt gta gat gaa ctc atg act gta ctc tac ccagaa tat 242 Ser Val Ser Ser Val Asp Glu Leu Met Thr Val Leu Tyr Pro GluTyr 20 25 30 tgg aaa atg tac aag tgt cag cta agg aaa gga ggc tgg caa cataac 290 Trp Lys Met Tyr Lys Cys Gln Leu Arg Lys Gly Gly Trp Gln His Asn35 40 45 aga gaa cag gcc aac ctc aac tca agg aca gaa gag act ata aaa ttt338 Arg Glu Gln Ala Asn Leu Asn Ser Arg Thr Glu Glu Thr Ile Lys Phe 5055 60 gct gca gca cat tat aat aca gag atc ttg aaa agt att gat aat gag386 Ala Ala Ala His Tyr Asn Thr Glu Ile Leu Lys Ser Ile Asp Asn Glu 6570 75 tgg aga aag act caa tgc atg cca cgg gag gtg tgt ata gat gtg ggg434 Trp Arg Lys Thr Gln Cys Met Pro Arg Glu Val Cys Ile Asp Val Gly 8085 90 95 aag gag ttt gga gtc gcg aca aac acc ttc ttt aaa cct cca tgt gtg482 Lys Glu Phe Gly Val Ala Thr Asn Thr Phe Phe Lys Pro Pro Cys Val 100105 110 tcc gtc tac aga tgt ggg ggt tgc tgc aat agt gag ggg ctg cag tgc530 Ser Val Tyr Arg Cys Gly Gly Cys Cys Asn Ser Glu Gly Leu Gln Cys 115120 125 atg aac acc agc acg agc tac ctc agc aag acg tta ttt gaa att aca578 Met Asn Thr Ser Thr Ser Tyr Leu Ser Lys Thr Leu Phe Glu Ile Thr 130135 140 gtg cct ctc tct caa ggc ccc aaa cca gta aca atc agt ttt gcc aat626 Val Pro Leu Ser Gln Gly Pro Lys Pro Val Thr Ile Ser Phe Ala Asn 145150 155 cac act tcc tgc cga tgc atg tct aaa ctg gat gtt tac aga caa gtt674 His Thr Ser Cys Arg Cys Met Ser Lys Leu Asp Val Tyr Arg Gln Val 160165 170 175 cat tcc att att aga cgt tcc ctg cca gca aca cta cca cag tgtcag 722 His Ser Ile Ile Arg Arg Ser Leu Pro Ala Thr Leu Pro Gln Cys Gln180 185 190 gca gcg aac aag acc tgc ccc acc aat tac atg tgg aat aat cacatc 770 Ala Ala Asn Lys Thr Cys Pro Thr Asn Tyr Met Trp Asn Asn His Ile195 200 205 tgc aga tgc ctg gct cag gaa gat ttt atg ttt tcc tcg gat gctgga 818 Cys Arg Cys Leu Ala Gln Glu Asp Phe Met Phe Ser Ser Asp Ala Gly210 215 220 gat gac tca aca gat gga ttc cat gac atc tgt gga cca aac aaggag 866 Asp Asp Ser Thr Asp Gly Phe His Asp Ile Cys Gly Pro Asn Lys Glu225 230 235 ctg gat gaa gag acc tgt cag tgt gtc tgc aga gcg ggg ctt cggcct 914 Leu Asp Glu Glu Thr Cys Gln Cys Val Cys Arg Ala Gly Leu Arg Pro240 245 250 255 gcc agc tgt gga ccc cac aaa gaa cta gac aga aac tca tgccag tgt 962 Ala Ser Cys Gly Pro His Lys Glu Leu Asp Arg Asn Ser Cys GlnCys 260 265 270 gtc tgt aaa aac aaa ctc ttc ccc agc caa tgt ggg gcc aaccga gaa 1010 Val Cys Lys Asn Lys Leu Phe Pro Ser Gln Cys Gly Ala Asn ArgGlu 275 280 285 ttt gat gaa aac aca tgc cag tgt gta tgt aaa aga acc tgcccc aga 1058 Phe Asp Glu Asn Thr Cys Gln Cys Val Cys Lys Arg Thr Cys ProArg 290 295 300 aat caa ccc cta aat cct gga aaa tgt gcc tgt gaa tgt acagaa agt 1106 Asn Gln Pro Leu Asn Pro Gly Lys Cys Ala Cys Glu Cys Thr GluSer 305 310 315 cca cag aaa tgc ttg tta aaa gga aag aag ttc cac cac caaaca tgc 1154 Pro Gln Lys Cys Leu Leu Lys Gly Lys Lys Phe His His Gln ThrCys 320 325 330 335 agc tgt tac aga cgg cca tgt acg aac cgc cag aag gcttgt gag cca 1202 Ser Cys Tyr Arg Arg Pro Cys Thr Asn Arg Gln Lys Ala CysGlu Pro 340 345 350 gga ttt tca tat agt gaa gaa gtg tgt cgt tgt gtc ccttca tat tgg 1250 Gly Phe Ser Tyr Ser Glu Glu Val Cys Arg Cys Val Pro SerTyr Trp 355 360 365 caa aga cca caa atg agc taagattgta ctgttttccagttcatcgat 1298 Gln Arg Pro Gln Met Ser 370 tttctattat ggaaaactgtgttgccacag tagaactgtc tgtgaacaga gagacccttg 1358 tgggtccatg ctaacaaagacaaaagtctg tctttcctga accatgtgga taactttaca 1418 gaaatggact ggagctcatctgcaaaaggc ctcttgtaaa gactggtttt ctgccaatga 1478 ccaaacagcc aagattttcctcttgtgatt tctttaaaag aatgactata taatttattt 1538 ccactaaaaa tattgtttctgcattcattt ttatagcaac aacaattggt aaaactcact 1598 gtgatcaata tttttatatcatgcaaaata tgtttaaaat aaaatgaaaa ttgtatttat 1658 aaaaaaaaaa aaaaaa 16742 419 PRT Homo sapiens 2 Met His Ser Leu Gly Phe Phe Ser Val Ala Cys SerLeu Leu Ala Ala -45 -40 -35 Ala Leu Leu Pro Gly Pro Arg Glu Ala Pro AlaAla Ala Ala Ala Phe -30 -25 -20 -15 Glu Ser Gly Leu Asp Leu Ser Asp AlaGlu Pro Asp Ala Gly Glu Ala -10 -5 -1 1 Thr Ala Tyr Ala Ser Lys Asp LeuGlu Glu Gln Leu Arg Ser Val Ser 5 10 15 Ser Val Asp Glu Leu Met Thr ValLeu Tyr Pro Glu Tyr Trp Lys Met 20 25 30 Tyr Lys Cys Gln Leu Arg Lys GlyGly Trp Gln His Asn Arg Glu Gln 35 40 45 50 Ala Asn Leu Asn Ser Arg ThrGlu Glu Thr Ile Lys Phe Ala Ala Ala 55 60 65 His Tyr Asn Thr Glu Ile LeuLys Ser Ile Asp Asn Glu Trp Arg Lys 70 75 80 Thr Gln Cys Met Pro Arg GluVal Cys Ile Asp Val Gly Lys Glu Phe 85 90 95 Gly Val Ala Thr Asn Thr PhePhe Lys Pro Pro Cys Val Ser Val Tyr 100 105 110 Arg Cys Gly Gly Cys CysAsn Ser Glu Gly Leu Gln Cys Met Asn Thr 115 120 125 130 Ser Thr Ser TyrLeu Ser Lys Thr Leu Phe Glu Ile Thr Val Pro Leu 135 140 145 Ser Gln GlyPro Lys Pro Val Thr Ile Ser Phe Ala Asn His Thr Ser 150 155 160 Cys ArgCys Met Ser Lys Leu Asp Val Tyr Arg Gln Val His Ser Ile 165 170 175 IleArg Arg Ser Leu Pro Ala Thr Leu Pro Gln Cys Gln Ala Ala Asn 180 185 190Lys Thr Cys Pro Thr Asn Tyr Met Trp Asn Asn His Ile Cys Arg Cys 195 200205 210 Leu Ala Gln Glu Asp Phe Met Phe Ser Ser Asp Ala Gly Asp Asp Ser215 220 225 Thr Asp Gly Phe His Asp Ile Cys Gly Pro Asn Lys Glu Leu AspGlu 230 235 240 Glu Thr Cys Gln Cys Val Cys Arg Ala Gly Leu Arg Pro AlaSer Cys 245 250 255 Gly Pro His Lys Glu Leu Asp Arg Asn Ser Cys Gln CysVal Cys Lys 260 265 270 Asn Lys Leu Phe Pro Ser Gln Cys Gly Ala Asn ArgGlu Phe Asp Glu 275 280 285 290 Asn Thr Cys Gln Cys Val Cys Lys Arg ThrCys Pro Arg Asn Gln Pro 295 300 305 Leu Asn Pro Gly Lys Cys Ala Cys GluCys Thr Glu Ser Pro Gln Lys 310 315 320 Cys Leu Leu Lys Gly Lys Lys PheHis His Gln Thr Cys Ser Cys Tyr 325 330 335 Arg Arg Pro Cys Thr Asn ArgGln Lys Ala Cys Glu Pro Gly Phe Ser 340 345 350 Tyr Ser Glu Glu Val CysArg Cys Val Pro Ser Tyr Trp Gln Arg Pro 355 360 365 370 Gln Met Ser 3196 PRT Homo sapiens 3 Met Arg Thr Leu Ala Cys Leu Leu Leu Leu Gly CysGly Tyr Leu Ala 1 5 10 15 His Val Leu Ala Glu Glu Ala Glu Ile Pro ArgGlu Val Ile Glu Arg 20 25 30 Leu Ala Arg Ser Gln Ile His Ser Ile Arg AspLeu Gln Arg Leu Leu 35 40 45 Glu Ile Asp Ser Val Gly Ser Glu Asp Ser LeuAsp Thr Ser Leu Arg 50 55 60 Ala His Gly Val His Ala Thr Lys His Val ProGlu Lys Arg Pro Leu 65 70 75 80 Pro Ile Arg Arg Lys Arg Ser Ile Glu GluAla Val Pro Ala Val Cys 85 90 95 Lys Thr Arg Thr Val Ile Tyr Glu Ile ProArg Ser Gln Val Asp Pro 100 105 110 Thr Ser Ala Asn Phe Leu Ile Trp ProPro Cys Val Glu Val Lys Arg 115 120 125 Cys Thr Gly Cys Cys Asn Thr SerSer Val Lys Cys Gln Pro Ser Arg 130 135 140 Val His His Arg Ser Val LysVal Ala Lys Val Glu Tyr Val Arg Lys 145 150 155 160 Lys Pro Lys Leu LysGlu Val Gln Val Arg Leu Glu Glu His Leu Glu 165 170 175 Cys Ala Cys AlaThr Thr Ser Leu Asn Pro Asp Tyr Arg Glu Glu Asp 180 185 190 Thr Asp ValArg 195 4 241 PRT Homo sapiens 4 Met Asn Arg Cys Trp Ala Leu Phe Leu SerLeu Cys Cys Tyr Leu Arg 1 5 10 15 Leu Val Ser Ala Glu Gly Asp Pro IlePro Glu Glu Leu Tyr Glu Met 20 25 30 Leu Ser Asp His Ser Ile Arg Ser PheAsp Asp Leu Gln Arg Leu Leu 35 40 45 His Gly Asp Pro Gly Glu Glu Asp GlyAla Glu Leu Asp Leu Asn Met 50 55 60 Thr Arg Ser His Ser Gly Gly Glu LeuGlu Ser Leu Ala Arg Gly Arg 65 70 75 80 Arg Ser Leu Gly Ser Leu Thr IleAla Glu Pro Ala Met Ile Ala Glu 85 90 95 Cys Lys Thr Arg Thr Glu Val PheGlu Ile Ser Arg Arg Leu Ile Asp 100 105 110 Arg Thr Asn Ala Asn Phe LeuVal Trp Pro Pro Cys Val Glu Val Gln 115 120 125 Arg Cys Ser Gly Cys CysAsn Asn Arg Asn Val Gln Cys Arg Pro Thr 130 135 140 Gln Val Gln Leu ArgPro Val Gln Val Arg Lys Ile Glu Ile Val Arg 145 150 155 160 Lys Lys ProIle Phe Lys Lys Ala Thr Val Thr Leu Glu Asp His Leu 165 170 175 Ala CysLys Cys Glu Thr Val Ala Ala Ala Arg Pro Val Thr Arg Ser 180 185 190 ProGly Gly Ser Gln Glu Gln Arg Ala Lys Thr Pro Gln Thr Arg Val 195 200 205Thr Ile Arg Thr Val Arg Val Arg Arg Pro Pro Lys Gly Lys His Arg 210 215220 Lys Phe Lys His Thr His Asp Lys Thr Ala Leu Lys Glu Thr Leu Gly 225230 235 240 Ala 5 232 PRT Homo sapiens 5 Met Asn Phe Leu Leu Ser Trp ValHis Trp Ser Leu Ala Leu Leu Leu 1 5 10 15 Tyr Leu His His Ala Lys TrpSer Gln Ala Ala Pro Met Ala Glu Gly 20 25 30 Gly Gly Gln Asn His His GluVal Val Lys Phe Met Asp Val Tyr Gln 35 40 45 Arg Ser Tyr Cys His Pro IleGlu Thr Leu Val Asp Ile Phe Gln Glu 50 55 60 Tyr Pro Asp Glu Ile Glu TyrIle Phe Lys Pro Ser Cys Val Pro Leu 65 70 75 80 Met Arg Cys Gly Gly CysCys Asn Asp Glu Gly Leu Glu Cys Val Pro 85 90 95 Thr Glu Glu Ser Asn IleThr Met Gln Ile Met Arg Ile Lys Pro His 100 105 110 Gln Gly Gln His IleGly Glu Met Ser Phe Leu Gln His Asn Lys Cys 115 120 125 Glu Cys Arg ProLys Lys Asp Arg Ala Arg Gln Glu Lys Lys Ser Val 130 135 140 Arg Gly LysGly Lys Gly Gln Lys Arg Lys Arg Lys Lys Ser Arg Tyr 145 150 155 160 LysSer Trp Ser Val Tyr Val Gly Ala Arg Cys Cys Leu Met Pro Trp 165 170 175Ser Leu Pro Gly Pro His Pro Cys Gly Pro Cys Ser Glu Arg Arg Lys 180 185190 His Leu Phe Val Gln Asp Pro Gln Thr Cys Lys Cys Ser Cys Lys Asn 195200 205 Thr Asp Ser Arg Cys Lys Ala Arg Gln Leu Glu Lys Asn Glu Arg Thr210 215 220 Cys Arg Cys Asp Lys Pro Arg Arg 225 230 6 14 PRT Homosapiens 6 Pro Xaa Cys Val Xaa Xaa Xaa Arg Cys Xaa Gly Cys Cys Asn 1 5 107 50 DNA Homo sapiens 7 tgtaatacga ctcactatag ggatcccgcc atggaggccacggcttatgc 50 8 28 DNA Homo sapiens 8 gatctctaga ttagctcatt tgtggtct 289 27 DNA Homo sapiens 9 cgcggatcca tgactgtact ctaccca 27 10 60 DNA Homosapiens 10 cgctctagat caagcgtagt ctgggacgtc gtatgggtac tcgaggctcatttgtggtct 60

What is claimed:
 1. An isolated polynucleotide comprising a memberselected from the group consisting of: (a) a polynucleotide encoding thepolypeptide as set forth in SEQ ID NO: 2; (b) a polynucleotide capableof hybridizing to and which is at least 70% identical to thepolynucleotide of (a); and (c) a polynucleotide fragment of thepolynucleotide of (a) or (b).
 2. The polynucleotide of claim 1, whereinthe polynucleotide is DNA.
 3. The polynucleotide of claim 2, whichencodes the polypeptide as set forth in SEQ ID NO:
 2. 4. Thepolynucleotide of claim 2, which encodes the polypeptide comprising −46to 373 as set forth in SEQ ID NO:
 2. 5. The polynucleotide of claim 2,which encodes the polypeptide comprising 1 to 373 as set forth in SEQ IDNO:
 2. 6. An isolated polynucleotide comprising a member selected fromthe group consisting of (a) a polynucleotide which encodes a maturepolypeptide encoded by the DNA contained in ATCC Deposit No. 97149; (b)a polynucleotide which encodes a polypeptide expressed by the DNAcontained in ATCC Deposit No. 97149; (c) a polynucleotide capable ofhybridizing to and which is at least 70% identical to the polynucleotideof (a) or (b); and (d) a polynucleotide fragment of the polynucleotideof (a), (b) or (c).
 7. A vector containing the DNA of claim
 2. 8. A hostcell genetically engineered with the vector of claim
 7. 9. A process forproducing a polypeptide comprising: expressing from the host cell ofclaim 8 the polypeptide encoded by said DNA.
 10. A process for producingcells capable of expressing a polypeptide comprising transforming ortransfecting the cells with the vector of claim
 7. 11. A polypeptideselected from the group consisting of: (i) a polypeptide having thededuced amino acid sequence of SEQ ID NO: 2 and fragments, analogs andderivatives thereof; (ii) a polypeptide comprising amino acid 1 to aminoacid 373 of SEQ ID NO: 2; and (iii) a polypeptide encoded by the cDNA ofATCC Deposit No. 97149 and fragments, analogs and derivatives of saidpolypeptide.
 12. A compound effective as an agonist for the polypeptideof claim
 11. 13. A compound effective as an antagonist against thepolypeptide of claim
 11. 14. A method for the treatment of a patienthaving need of VEGF-2 comprising administering to the patient atherapeutically effective amount of the polypeptide of claim
 11. 15. Themethod of claim 14, wherein said therapeutically effective amount of thepolypeptide is administered by providing to the patient DNA encodingsaid polypeptide and expressing said polypeptide in vivo.
 16. A methodfor the treatment of a patient having need of VEGF-2 comprising:administering to the patient a therapeutically effective amount of thecompound of claim
 12. 17. A method for the treatment of a patient havingneed to inhibit VEGF-2 comprising: administering to the patient atherapeutically effective amount of the antagonist of claim
 13. 18. Aprocess for diagnosing a disease or a susceptibility to a diseaserelated to expression of the polypeptide of claim 11 comprisingdetermining a mutation in the nucleic acid sequence encoding saidpolypeptide.
 19. A diagnostic process comprising analyzing for thepresence of the polypeptide of claim 11 in a sample derived from a host.20. A method for identifying compounds which bind to and activate orinhibit a receptor or the polypeptide of claim 11 comprising: (a)contacting a cell expressing on the surface thereof a receptor for thepolypeptide, said receptor being associated with a second componentcapable of providing a detectable signal in response to the binding of acompound to said receptor, with a compound to be screened underconditions to permit binding to the receptor; and (b) determiningwhether the compound binds to and activates or inhibits the receptor bydetecting the presence or absence of a signal generated from theinteraction of the compound with the receptor.